Pullulanase is a debranching enzyme which can be used in the brewing industry to make low calorie beer and in the beverage industry to make high dextrose syrups. See, for example, U.S. Pat. Nos. 4,355,110 and 4,355,047. These patents, as well as any other patents and/or references hereinafter referred to, are hereby incorporated by reference as if fully set forth herein.
The pullulanase gene has been isolated, sequenced and characterized from bacterial organisms. For example, see, Kuriki, et al., 170 J. Bacteriology, 1554 (1988). Rice and other grains have been known to contain pullulanase. For instance, U.S. Pat. No. 4,355,110 discloses the presence of pullulanase in rice.
The pullulanase enzyme can be isolated from rice by the method disclosed in U.S. Pat. No. 4,355,110. One problem with this approach, however, is that a great deal of waste byproduct is generated. One is therefore faced with disposal problems associated with this waste.
Another alternative source of pullulanase is from bacterial cultures. However, the use of bacteria may have certain negative connotations with the public. Also, bacterial pullulanase is generally less active than rice pullulanase.
Accordingly, there is a need for an alternative supply of rice pullulanase enzyme for use in making low calorie beer or high dextrose syrups. The present invention overcomes the aforementioned problems in providing a yeast that is made to express, properly process, and secrete the rice pullulanase enzyme.
Yeast is considered to be a better host organism for the production of food processing ingredients because it is generally regarded as safe and it can be made to express, properly process and secrete certain heterologous proteins. The problem is that some proteins cannot be produced in yeast (for example, some are toxic) and others cannot be properly processed and/or secreted. Each protein must be handled on a case-by-case basis with the probability of success impossible to predict a priori.
The present invention overcomes these problems by providing an expression construct that is capable of directing the expression of a mature pullulanase enzyme in yeast. The invention is more surprising in that the construct expresses an enzyme that does not mimic the natural rice pullulanase amino acid sequence.
The phrase "mature pullulanase" refers to the pullulanase isolated from rice seed. In the mature pullulanase the methionine or a peptide containing the methionine is assumed to have been removed during post translational modification. However, the mRNA sequence must have a methionine residue encoded since it is the translation initiation codon. This is one of the problems that had to be overcome when expressing the pullulanase enzyme in a yeast system as in the present invention.